Concluding remarks

Our results show that the five centuries of contact and admixture among the populations meeting in America has produced complex and varied population structure and co-ancestry patterns amongst all the populations we studied, especially in non-admixed indigenous peoples. Despite a severe reduction in indigenous

American population sizes during this time, American co-ancestry persists in varied degrees in contemporary populations across the continent. This indigenous American gene pool has also survived in places where it might be expected to have been reduced by a strong influx of Europeans - notably in the urban samples we analyzed from Guatemala City, Chile and Mexicans in Los Angeles.

The PIMA panel’s capacity to differentiate American and East Asian populations to a level equivalent to, or better than, a much larger genomic ancestry control panel comprising 314 SNPs, exceeded our expectations for the goal of adjusting the shortfall of the 34plex panel that had been identified from initial comparisons of HGDP-CEPH Americans to other populations. A combined PIMA+34plex panel has a more balanced informativeness content and can identify and differentiate American-indicative variants, even when present in relatively minor proportions in admixed individuals. This is substantiated by the detection of all four population group components in the complex admixture patterns of the São Paulo sample, with levels of American and East Asian co-ancestry below 5%. We recognize that inclusion of the seven SNPs in a full 34plex panel, absent due to poor performance when testing study populations, is likely to boost the ancestry differentiation performance of the PIMA+34plex sets still further, notably the most powerful East Asian-informative SNP in 34plex: rs3827760 [16]. Nevertheless, the small scale and high differentiating power of PIMA (particularly in combination with 34plex) makes it an invaluable and practical tool for population genetics, medical studies and forensic DNA analysis.

Acknowledgments

The authors would like to thank the populations who kindly agreed to participate in the study and field teams who made sample collection possible. AFA is supported by a post-doctorate grant funded by the Consellería de Cultura, Educación e Ordenación Universitaria e da Consellería de Economía, Emprego e Industria from Xunta de Galicia, Spain (Modalidade B, ED481B 2018/010). CCG was supported by a doctoral scholarship funded by CNPq.

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Figure captions

Fig. 1. Map of American study population and HGDP-CEPH reference population sampling locations.

Fig. 2. Summary bar-plots of PIMA SNP variation in the four main population groups of 1000 Genomes (1KG) and HGDP-CEPH indigenous Americans. SNPs are ordered left to right in descending allele frequency differential values (delta) comparing the 1KG 4-group average allele frequencies (non-AME) vs CEPH American (AME) frequencies (not calculated for the tri-allelic SNP rs17287498 and X-SNP rs3027749). The non-American allele frequencies of rs17287498 were estimated from gnomAD population data (lacking SAS). Additional plots shown for the 1KG 4-group average frequencies and 1KG Peruvians from Lima (PEL). RA: reference allele; EUR: Europeans; AFR: Africans; SAS: South Asian; EAS: East Asian.

Fig. 3. Evaluation of the population differentiation performance of individual PIMA and 34plex panels and the combined set. Fig. 3A. PCA tests of 3 SNP sets analyzing 1000 Genomes African, European, East Asian; and HGDP-CEPH American genotype data. Fig. 3B. Cumulative I _n charts of PCA tests of 3 SNP sets, adding most powerful loci first. Fig. 3C. Cross validation ancestry assignment success of 3 SNP sets, with error highlighted for the relevant population group.

Fig. 4A. STRUCTURE cluster plots for K:3 and K:4 inferred clusters comparing PIMA+34plex and LACE panels for a comprehensive set of common reference and American study samples. Cluster bar plots ordered in both SNP panels by increasing majority co-ancestry from LACE cluster membership proportions (CLUMPP-merged from 5 runs). HGDP-CEPH populations grouped and marked as: A=Amazonian Karitiana /Surui; M=Maya; P=Pima. Fig. 4B. Cross validation ancestry assignment

rates for the reference population samples. Fig. 4C. Comparisons of mean population differentiation metrics F _ST and I _n in each panel.

Fig. 5. PCAs of American study population subsets arranged in four plots. (A) Indigenous American study populations (Colombia: Awa, Pastos, Embera, Pijao, Coyaima; Venezuela: Wayu; Guatemala: Q’eqchi). (B) American admixed populations with high European ancestry (Colombia: CLM, NW Colombia, Bucaramanga; Venezuela: Caracas, Maracaibo; Brazil: São Paulo; Chile: North and South; MXL; PUR). (C) American admixed populations with high African ancestry (Brazil: Kalunga, Sacutiaba; Colombia: Mulaló; ASW). (D) Urban populations with high American ancestry (MXL, Guatemala City, and north/south Chile).